All Things Chromatography Blog

Category Archives: TLC Plates

Have you ever pulled your TLC plate out of the developing tank, only to realize that your Rfs are out of whack? Or the analyte you were expecting to see is missing or still overlapping another component? Time is wasted. Emotions flare. Did you forget something? Are the plates any good? Did you pick the wrong profession?

Many factors affect your results in chromatography. They include:

layer thickness

particle size and pore size of adsorbent

activation of adsorbent

mobile phase (quantity and freshness)

equilibrium inside the developing chamber

sample (size and application technique)

accurate Rf measurement

All of these controllable variables are important. To save time, you may be tempted to focus on the most influential and leave the others alone. However, some applications are more sensitive to slight variations. It’s better to invest time in the beginning to make sure every variable is just right. You’ll save time and energy—and will rest assured you’re indeed in the right profession.

That’s why we’re publishing a series of articles to explain these contributing factors in detail. (We know how much our analytical audience loves details!) Our goal is to broaden your overall knowledge in chromatography and help you achieve more consistent results.

In our last article, we described the first four of eight factors in choosing a TLC plate. Surely, you’ve been waiting with bated breath for the last four factors—and we don’t blame you! In this article, let’s look at these final considerations. Hold onto your seats…

Fluorescent indicator – UV254

This is an additive to the adsorbent which you may need depending on how your samples are visualized after the chromatography process. Present in only 2-4% by weight, this special material makes the entire plate glow blue or green (pretty!) when exposed to short wave, 254 nm UV light.

Why are fluorescent indicators important? Most sample types separated by TLC are not visible to the naked eye. This is usually because of the relatively small sample size or because there is no inherent coloring or shading in the compound. That’s why another method is needed to locate the sample components.

Some materials have an excited state that can be seen when exposed to 366 nm UV light. In this case, the components will fluoresce a characteristic color. In some cases, you need to spray the TLC plate with a solution before exposing it to UV light. The sample components will then be temporarily or permanently visible.

Back to UV 254 plates: Many compounds can absorb light around 254 nm. These include most compounds with aromatic rings or conjugated double bonds and some unsaturated compounds. When exposed to 254 nm UV light, absorbing compounds will fade (quench) the uniform fluorescence of the TLC plate, thus showing as dark spots on the otherwise bright background. This method is popular because it doesn’t modify or destroy the compounds, so it’s suitable for preparative TLC.

Glass plate scoring

The most popular size of TLC plate is 20×20 cm. Just as in life, being popular has its advantages; for TLC plates it means they’re usually the best value. Adding to that value, glass-backed TLC plates may be purchased in a pre-scored format. A pattern of scoring is etched onto the backs of plates, allowing you to “snap” them into smaller sizes as needed. Therefore, a pre-scored 20×20 cm plate can be snapped into four 5×20 cm or two 10×20 cm plates (kind of like Select-A-Size™ paper towels). It would cost you a lot more to purchase these same plates already in the smaller size. Other commonly ordered plates are the pre-scored 10×20 cm plates; they are popular for method development. They can be snapped into eight slide-sized plates (2.5×10 cm).

Preadsorbent zone

The preadsorbent zone is typically a non-reactive material coated adjacent to the regular TLC layer at the bottom of the plate. This special zone has two great properties. First, it allows you to apply a larger than average sample since it effectively compresses the sample into a tight “band” before entering the regular separation zone. This helps with separation power by improving vertical resolution of closely chromatographing sample components. Second, it allows you to apply samples quickly, without strict attention to the vertical location in the preadsorbent zone. In other words, the preadsorbent zone cleans up sloppy sample application. Note that you should always apply samples above the level of the mobile phase.

Channeling

Channeled TLC plates have adsorbent tracks (sample lanes) that are separated by channels where the adsorbent layer is removed from the glass backing. These specially made plates limit the horizontal spreading of sample components during development, thus preventing cross-contamination. In general, unless an adsorbent is coated in a way that causes a non-uniform layer, samples will always travel vertically and should never cross-contaminate an adjacent sample lane. However, for Type-A folks who crave absolute certainty—or in cases where clear evidence is needed for court—channeled plates are the way to go.

There you have it. You now know the eight key factors in selecting a TLC plate—you should feel like a pro now! Of course, if you need any additional guidance, our friendly team is here to help you.

“I’d like a box of TLC plates, please.” Sounds innocent enough, right? But when you have more than 500 plate options, things get a whole lot trickier. How do you dig through the layers (pun intended) to get the plates you need?

All clear now? Didn’t think so! In this first article of our two-part series, let’s look at the first four factors to help you make the right choice.

Adsorbent material
Most often, the actual layer is silica gel, but it can also be aluminum oxide, cellulose, microcrystalline cellulose, Florisil®, as well as various forms of modified silica gel such as reversed phase layers (C2, C8, C18), amino, cyano, and others. Which layer should you use? It depends on the compound you’re separating. Your best bet is to check what others have done first so you’re not reinventing the wheel. Historical data matters!

Plate backing
The layer’s backing is most often glass, because it can tolerate the most chromatography solvents. Using a flexible backing—namely polyethylene or aluminum—makes sense only when you must quickly and easily cut the plate (or sheet) into smaller sized pieces. Glass can also be cut or purchased pre-scored, so really it comes down to preference, availability, and price.

Layer thickness
Thickness depends on the chromatographic separation you’re doing. For instance, the thickness for standard analytical TLC is 250um (1/4th of a millimeter). But for quantitative level analytical TLC, you’ll need a thinner plate—typically 200um, 150um, or even 100um. This category of TLC has a special name: high performance thin layer chromatography, or HPTLC. Meanwhile, thicker plates (500um, 1000um, 1500um, and 2000um) are best for preparative applications where you’re separating much larger samples. In this case, you’ll want to recover the separated sample components with a scraper or suction device.

Plate size
This is a lot easier to figure out—whew! You just need to know about how many samples you want to run at the same time and what distance you need to chromatograph your samples for adequate separation. Standard sizes are 20×20, 10×20, 5×20, 10×10, 5×10, and 2.5×7.5 (microscope slide). Of course, all measurements are in centimeters. Additional specialty sizes are 2.5×10, 5×5, 2.5×5, and, our favorite, 20×40. Yes, you do need a very large custom developing tank for that one!

We hope this gets you on the right track when you need TLC plates. For more info, stay tuned for part II, where we explain fluorescent indicator, scored plates, preadsorbent zone, and channeling. We find this stuff so exciting and can’t wait to share it with you!

Since 1976, the National Council of Teachers of Mathematics has marked the week of October 10 as Metric Week (Oct. 10 = 10/10).

This year is especially nice, with the date being 10/10/10.

Once in a great while, we get requests for 8 x 8 thin layer chromatography plates – and that’s when we are reminded that not everyone has adopted the metric system (aka “international system of units” or “SI”).

The whole concept of a decimal-based system of measurements was first proposed in 1585 by Simon Stevin in his book, “The Tenth.”

The idea has been embraced by great minds like Thomas Jefferson, and the U.S. Mint produced the world’s first decimal currency in 1792.

Click Here for more interesting facts surrounding the history of the Metric System.

Click Here for more on Metric Week from the National Institute of Standards and Technology (NIST).

By the way, we won’t say anything bad about you if you order 8 x 8 plates, just don’t be surprised when you get them and they say “20 x 20” on the side of the box.

We picked up this application of Thin Layer Chromatography from MyPlainview.com:

While they haven’t exactly discovered a cure for cancer, what Hughes and Arauza did over the summer months does have quite a bit of value — both in terms of scientific research and in their own personal education and edification…

The pair technically started their research in the spring 2009 term after learning in November that they were chosen for the program. They spent the spring doing an extensive literature review once they chose a topic from the umbrella of choices they were presented.

They chose to follow a path started by May 2009 graduate Joanne Jacob, who had experimented with 12 different herbs and their effect on tumor growth in mice. One in particular had significant results in Jacob’s research, and the two coeds decided to further check out Ashwagandha, commonly known as Indian Ginseng and used by many to treat depression, inflammation and neurological disorders.

Using a powdered form of the root, Hughes and Arauza first rinsed it to remove any lipids, then ran a 6-hour process known as a Soxhlet to liquify the extract into a more usable form. Gray likened the process to a drip coffee maker, where heated water (or in this case methanol) runs through the extract and then back through repeatedly until it is complete.

Using thin-layer chromatography on glass plates, the team was able to separate the extract into various compounds. Through nearly 30 plates — a time-consuming process itself — the duo was able to identify one particular compound that was strong every time. They eluded the compound from the plate and tested it on 4T1 breast cancer cells grown in Petri dishes to determine how it would affect the cells.

The results were astonishing.

“This was really annihilating the breast cancer cells,” noted Arauza, pointing to a chart of the results that showed the cell growth was dramatically reduced compared to even the full extract. “This one was very potent; none of the others were even close.”

Jennifer Bachman, Laboratory Coordinator and Inventory Specialist for the Chemistry Department at Sewanee: The University of the South, talks about how satisfied everyone at Sewanee is with our Uniplate Thin Layer Chromatography plates.